Blood coagulation is a host defense mechanism provoked in response to vascular injury and/or foreign stimulation. Blood coagulation involves 15 factors including 12 proteinaceous coagulation factors in plasma, along with calcium ion, tissue factor and phospholipid (platelet-derived). This reaction is mediated by a cascade mechanism, in which a series of protease activations occurs successively on the membrane of platelets aggregated at a site of injury or damaged endothelial cells.
The blood coagulation cascade is divided into intrinsic and extrinsic pathways. It is called extrinsic blood coagulation when it occurs with the aid of tissue factor present in tissues, while it is called intrinsic blood coagulation when it occurs without the aid of tissue factor.
Intrinsic blood coagulation is initiated by the contact of blood coagulation factor XII in plasma with the surface of a negatively-charged solid phase or the like. Upon adsorption onto the surface, factor XII is converted through limited hydrolysis into activated factor XII (XIIa), an active protease. In turn, factor XIIa causes the limited hydrolysis of factor XI into activated factor XI (XIa), an active protease. After such a cascade of protease activations, the final protease thrombin causes the limited hydrolysis of fibrinogen into fibrin, leading to the completion of blood coagulation. In downstream reactions after the activation of factor XI, a number of coagulation factors are assembled into complexes to facilitate coagulation factor localization at a site of hemostasis and to ensure efficient activation reactions. Namely, a tenase complex is assembled from phospholipids, factor VIIIa, factor IXa, factor X and Ca2+, while a prothrombinase complex is assembled from phospholipids, factor Va, factor Xa, prothrombin and Ca2+, resulting in significant promotion of prothrombin activation.
Extrinsic blood coagulation is initiated by the formation of a complex between factor VIIa and tissue factor. This complex between factor VIIa and tissue factor will join the intrinsic pathway at the stage of factor X and IX activation.
In general, extrinsic blood coagulation is reported to be important for hypercoagulation and physiological coagulation under pathological conditions.
Examples of known anticoagulants include a thrombin inhibitor such as heparin, as well as warfarin. However, since a thrombin inhibitor acts on downstream reactions of the blood coagulation cascade and hence cannot control the consumption of coagulation factors that lead to thrombin generation upon excess inhibition of coagulation, such a thrombin inhibitor involves a problem of hemorrhage tendency in clinical use. Likewise, warfarin inhibits the production of many blood coagulation factors and also involves a problem of hemorrhage tendency in clinical use, as in the case of a thrombin inhibitor.
As mentioned above, factor VIIa is located upstream in the extrinsic pathway and hence an inhibitor against factor VIIa will not affect the intrinsic coagulation pathway. That is, such an inhibitor will be able to leave the resistance against hemorrhage. This suggests that a factor VIIa inhibitor is expected to reduce the hemorrhage tendency, a side effect of existing anticoagulants. Thus, a factor VIIa inhibitor is expected to be effective in preventing or treating pathological conditions associated with the extrinsic coagulation pathway, e.g., chronic thrombosis (more specifically, postoperative deep vein thrombosis, post-PTCA restenosis, DIC (disseminated intravascular coagulation), cardioembolic strokes, cardiac infarction and cerebral infarction).
To date, some compounds have been reported as factor VIIa inhibitors (see, e.g., WO00/41531, WO00/35886, WO99/41231, EP921116A, WO00/15658, WO00/30646, WO00/58346).
However, all of these compounds are insufficient to have an inhibitory activity against factor VIIa or a selective inhibitory activity against extrinsic blood coagulation; there is a need to develop an agent having an improved inhibitory activity or an improved selective inhibitory activity.
Recent studies on enzyme inhibitors have tended to employ computational procedures, in which a three-dimensional enzyme model based on X-ray crystal structure analysis or the like is displayed on the screen of a computer to design a candidate compound which may have an inhibitory activity or to perform computer-aided virtual screening. Factor VIIa (hereinafter also referred to as “FVIIa”) has also been studied by X-ray structure analysis to determine its three-dimensional structure in free form, in complex with soluble tissue factor (this complex being hereinafter also referred to as “factor VIIa/soluble tissue factor” or “FVIIa/sTF), and in complex with a protein inhibitor (Nature, 380, 41-46, 1996; J. Mol. Biol, 285, 2089-2104, 1999; Proc Natl Acad Sci USA., 96, 8925-8930; J Struct Biol., 127, 213-223, 1999; Nature, 404, 465-470, 2000).
However, computational virtual docking techniques result in inaccurate estimation at present (Guidebook on Molecular Modeling Drug Design, 129-133, 1996, ACADEMIC PRESS); on the other hand, an enzyme molecule frequently undergoes an inhibitor brinding-induced conformational change called induced fit (Guidebook on Molecular Modeling Drug Design, 133-134, 1996, ACADEMIC PRESS). For computer-aided design of inhibitors, it is therefore most desirable to perform X-ray structure analysis on each inhibitor or its structurally similar inhibitor in complex with an enzyme to clarify the details of the binding mode between inhibitor and enzyme at the atomic level. In all previously reported crystals containing factor VIIa, however, irreversible inhibitors or protein inhibitors occupy the active sites of factor VIIa, which may be used as inhibitor-binding sites. Such crystals cannot be used for X-ray crystal structure analysis of a complex between factor VIIa and a low-molecular weight reversible inhibitor (e.g., having a molecular weight less than 1000). Generally, protein crystallization usually requires high purity. A problem of protease cleavage often arises in purifying such high-purity proteins (Crystallization of Nucleic Acids and Proteins, A Practical Approach, 34, 1992, IRL PRESS). In particular, a problem of self-cleavage arises in purifying and crystallizing a protease such as factor VIIa. For this reason, an irreversible inhibitor is often used in purification and crystallization because once binding occurs, the irreversible inhibitor will not be released from the protease and allows complete prevention of self-cleavage during purification and crystallization. However, in the case of a complex with a low-molecular weight reversible inhibitor, it involves technical difficulties because there is no guarantee that self-cleavage is completely prevented during crystallization. Indeed, there has been no report showing the crystallization or three-dimensional structure of a complex between factor VIIa and a low-molecular weight reversible factor VIIa inhibitor.